Rapid and ultra-sensitive detection of Staphylococcus aureus with aptamer-conjugated gold nanoparticles

Yi-Chung Chang, Chia-Ying Yang, Ruei-Lin Sun, Yi-Feng Cheng, Wei-Chen Kao,Pan-Chyr Yang *

Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan, 115, ROC

Supplementary figures and text:

Supplementary Figure 1: Flowchart for cell-based SELEX.
Supplementary Figure 2:A dendrogram analysis of 96 oligonucleotide sequences after 8 rounds of cell-based SELEX.
Supplementary Figure 3:Result of fluorescence microscopy of SA17 and SA61 against different bacteria.
Supplementary Figure 4: Dissociation constant (Kd) and predicted structures of SA17 and SA61.
Supplementary Figure 5:Binding capacity of SA17 and SA61 on S. aureus cells.
Supplementary Figure 6. Binding capacity of SA17 and SA61 on 60 nm GNPs.
Supplementary Figure 7:Characterization the light scattering signal of GNPs.
Supplementary Figure 8:Single bacteria detection by bead-based amplification.
Supplementary Table 1:The interaction of SA aptamers (SA17 and SA61) with 21 bacteria strains
Supplementary Table 2: Aptamer sequences in round 8 pool.

Supplementary Figure 1.Flowchart for cell-based SELEX. The selection process is based on hybridization of S. aureus with a library of DNA aptamers in solution followed by counter-selection using S. epidermidis.

Supplementary Figure 2.A dendrogram analysis of 96 oligonucleotide sequences after eight rounds of cell-based SELEX.

Supplementary Figure 3.Fluorescence microscopic detection of SA17 and SA61 binding to different bacteria. Biotin-labeled SA17 or SA61 (500 nM) were incubated with S. aureus cells at 4°C for 30 minutes and stained with streptavidin-PE. Paired sets of images for each of the tested bacterial strains incubated with SA17 (left pair) and SA61 (right pair) are shown. Left image in each pair: bright field; right image in each pair: fluorescence microscopy.

Supplementary Figure 4. Kds and predicted structures of SA17 and SA61. (A) Kd measured by a total binding assay based on quantification of bound SA17 (left) and SA61 (right) aptamers by qPCR. Different concentrations of aptamers were incubated with equal numbers of S. aureus cells; bound aptamers were eluted and quantified by qPCR. The calculated Kds of SA17 and SA61 for S. aureus binding were 35 and 129 nM, respectively. (B) Secondary structures predicted with mfold software for SA17 (left) and SA61 (right). ∆G values represent the stability of the structures. (C) Kds for SA17-GNPs (left) and SA61-GNPs (right) were determined by quantification of bound aptamer-GNPs. Different concentrations of aptamer-GNPs were incubated with equal numbers of S. aureus cells, and bound aptamer-GNPs were eluted and quantified. The Kds of SA17-GNPs and SA61-GNPs for S. aureuswere 3.03 and 9.9 nM, respectively.

Supplementary Figure 5. Binding capacity of S. aureus cells for SA17 and SA61. (A) SA17 (250 nM) was incubated with samples containing 10, 100, 1000, and 10,000 S.aureus cells; a no-cell control was included as a background signal for calculation of CT. After removing unbound aptamers, bound aptamers were eluted by heating and quantified by qPCR. The results are shown in the upper panel. The number of bound SA17 molecules per S. aureus cell is calculated from the CT by reference to the standard curve shown in the lower panel. (B) The results for SA61 aptamers obtained as in A.

Supplementary Figure 6. Binding capacity of 60 nm GNPs for SA17 and SA61. (A) Thio-adaptor sequences (5 M) were conjugated onto 60-nm GNPs, and adaptor-GNPs were further incubated with 5 M SA17 aptamer. After washing away the unbound aptamers, SA17-GNPs were quantified by measuring OD550. Different numbers of SA17-GNPs (10, 100, 1000, and 10,000) were analyzed by qPCR to calculate the amount of bound aptamer; buffer alone was used as a background control. (B) Binding capacity of GNPs for SA61 determined as in A.

Supplementary Figure 7. Characterization of the light-scattering signal of GNPs. a, Light-scattering signals from serially diluted samples of 60-nm GNPs. The highest concentration is 3105particles/l. The control is buffer alone. b, Light-scattering intensity of different sizes (15, 30, 60, and 100 nm) and concentrations of GNPs. The scattering results indicated that the signal is increased in proportion to particle size and concentration. The detection limit for 100, 60, 30, and 15 nm GNPs were 63  21, 508  176, 7.8104, and 5106 particles/l, respectively. Open circle: 100 nm GNPs; open square: 60 nm GNPs; triangle: 30 nm GNPs; inverted triangle: 15 nm GNPs. c, The light-scattering data fit to a nonlinear equation showing that signal intensity increases with the sixth power of the particle radius, consistent with previous findings27.

Supplementary Figure 8. Detection of a single bacterium by bead-based amplification. Bacterial suspensions containing 10 bacterial cells, estimated based on OD600 values, were divided equally into 30 samples. Biotin-SA61-GNPs and SA17-M270 beads were added to each sample followed by application of the bead-based amplification protocol. The signal intensity of each sample was measured as above. Four independent assays were performed, with stars marking positive wells containing bacteria. The three gray bars at right show the signal for no-bacteria controls, and the red dashed line indicates the highest signal intensity in the three controls. The signal intensities of wells above the red dashed line (positive wells) are marked with asterisks. In four independent assays, 12, 19, 8, and 18 positive wells were obtained.

Binomial Nomenclature / ATCC Number / SA17 / SA61
Bacillus subtilis / 21336 / — / —
Citrobacter freundii / 8090 / — / —
Escherichia coli / 43896 / — / —
Klebsiella pneumoniae / 13883 / — / —
Listeria monocytogenes / 19112 / — / —
Moraxella catarrhalis / 25238 / — / —
Pseudomonas aeruginosa / 27853 / — / +/-
Salmonella enterica / 13314 / — / —
Shigella boydii / 8700 / — / —
Shigella flexneri / 29903 / — / —
Staphylococcus aureus Strain 0 / 6538DR / + / +
Staphylococcus aureus Strain 1 / 6538P / + / +
Staphylococcus aureus Strain 2 / 12600 / + / +
Staphylococcus aureus Strain 3 / 25923 / + / +
Staphylococcus aureus Strain 4 / 29213 / + / +
Staphylococcus aureus Strain 5 / 6538 / + / +
Staphylococcus epidermidis / 155 / — / +/-
Staphylococcus haemolyticus / 29970 / — / —
Staphylococcus saprophyticus / 15305 / — / —
Streptococcus bovis / 43077 / — / —
Streptococcus pneumoniae / 6301 / — / —

Supplementary Table 1. The interactions of SA aptamers (SA17 and SA61) with 21 bacterial strains, including six S. aureus strains and 15 bacteria from other genera or species. Binding assays were performed using IFA/fluorescence microscopy. Scoring of binding-signal ratios of aptamers to S. aureus relative to negative controls in IFAs: “-“, ratio <1.5-fold; “+/-”, ratio = 2–3-fold; “+”, ratio > 3-fold.

Name / sequence
SA-1 / TCCCTACGGCGCTAACCCACTCCCCTCCACCGCTCCGACTCCGTCCGCCACCGTGCTACAAC
SA-2 / TCCCTACGGCGCTAACCTCACACCGACCTGCTTCCCCCCCCCGGCCGCCACCGTGCTACAAC
SA-3 / TCCCTACGGCGCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCACCGTGCTACAAC
SA-4 / TCCCTACGGCGCTAACCCCATCACCGCACCTCCCACCGACTCCCCTGCCACCGTGCTACAAC
SA-5 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-6 / TCCCTACGGCGCTAACCCACCGCTCTCGCCCAGCTCCTCTCCTGCCGCCACCGTGCTACAAC
SA-7 / TCCCTACGGCGCTAACCCCTCCTCCCACCTCGCCCAGTCCGCCTACGCCACCGTGCTACAAC
SA-8 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-9 / TCCCTACGTGATTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-10 / TCCCTACGGCGCTAACCAGCCACGTCCCGTCCACCCCGCCACCTCCGCCACCGTGCTACAAC
SA-11 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-12 / TCCCTACGGCGCTAACCCCTGCTCCCCCCACCGTGTCCTCGCCTACGCCACCGTGCTACAAC
SA-13 / TCCCTACGGCGCTAACCACCCCCCCGGACCCGCTCTCCTGCCACTCGCCACCGTGCTACAAC
SA-14 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-15 / TCCCTACGGCGCTAACCTCACATCACTCCCCTCACCGCTACCCACCGCCACCGTGCTACAAC
SA-16 / TCCCTACGGCGCTAACCCCTCCTCGTCACCCTGCGCTCCCACCTCCGCCACCGTGCTACAAC
SA-17 / TCCCTACGGCGCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCACCGTGCTACAAC
SA-18 / TCCCTACGGCGCTAACCCCCCGCTCCCACGCTCTGCCCTCCTACCCGCCACCGTGCTACAAC
SA-19 / TCCCTACGGCGCTAACCACTCCTCTCCCGCAGCGCTTCCACCTCCCGCCACCGTGCTACAAC
SA-20 / TCCCTACGGCGCTAACCTGCTCCTCCCCGGTCCTCCAGCCTCCACCGCCACCGTGCTACAAC
SA-21 / TCCCTACGGCGCTAACCACTCCAGCATCCACCCTCCAGCCAACCCCGCCACCGTGCTACAAC
SA-22 / TCCCTACGGCGCTAACCCCAGTCCCGTCTCCCACCACGCCCCAGCTGCCACCGTGGTAAAAA
SA-23 / TCCCTACGGCGCTAACCCCTCCCCAGCCATCCTCCGCCACTCCACCGCCACCGTGCTACAAC
SA-24 / TCCCTACGGCGCTAACCCACCACCACTCCTCTCACCACGCACTCCCGCCACCGTGCTACAAC
SA-25 / TCCCTACGGCGCTAACCCTCACCAGTCCCCCGTCCCTCTCCCGTCCGCCACCGTGCTACAAC
SA-26 / TCCCTACGGCGCTAACCCCACACTCCCCGTCACCGCTCCACCGCCAGCCACCGTGCTACAAC
SA-27 / TCCCTACGGCGCTAACCCCCAACCGTCAGCTCACCCCGTCCTCCCCGCCACCGTGCTACAAC
SA-28 / TCCCTACGGCGCTAACCCCTCCACCGAACCTCCCACGCTCCCCGCCGCCACCGTGCTACAAC
SA-29 / TCCCTACGGCGCTAACCCCCTGCCCACTCCACACCGTCACCACACCGCCACCGTGCTACAAC
SA-30 / TCCCTACGGCGCTAACCCCTCTCACCGGTCGTCCTCCCCACCTCCAGCCACCGTGCTACAAC
SA-31 / TCCCTACGGCGCTAACCCAGTCAACCTCCCCGTCCTCCCGCCAACGCCACCGTGCTACAAC
SA-32 / TCCCTACGGCGCTAACCCACTGTCCCCCGTCCCTCCGAGCCTCTCCGCCACCGTGCTACAAC
SA-33 / TCCCTACGGCGCTAACCCCTCCCGCTCACTCACCCGTCCCGCCTACGCCACCGTGCTACAAC
SA-34 / TCCCTACGGCGCTAACACGACAACCGCTACCCCGTCCAGCTCCCCCGCCACCGTGCTACAAC
SA-36 / TCCCTACGGCGCTAACCCCTCCACCGGCTCCCTCGCTACCCCACCCGCCACCGTGCTACAAC
SA-37 / TCCCTACGGCGCTAACCACTCCTCCCCGCCTGGCCACCGTGCTACAAC
SA-38 / TCCCTACGGCGCTAACCCCCACTCCTCCATCCCGTCGCCCTCCATCGCCACCGTGCTACAAC
SA-39 / TCCCTACGGCGCTAACCCTCCCCCCAGCTCCTCTCCACCTCGCCTGGCCACCGTGCTACAAC
SA-40 / TCCCTACGGCGCTAACCCCTCCTCACCCCGCGTCCTCCCACGTCTCGCCACCGTGCTACAAC
SA-41 / TCCCTACGGCGCTAACCCCTCCTCCCACCACAGACCGACTCCCCTCGCCACCGTGCTACAAC
SA-42 / TCCCTACGGCGCTAACCCCTCCTCCCACCACCAGCCGCTCAACTCCGCCACCGTGCTACAAC
SA-45 / TCCCTACGGCGCTAACCTCCTACCGTCCACCCCCACAGCTCCTCCGCCACCGTGCTACAAC
SA-46 / TCCCTACGGCGCTAACCCCCTCCGTCACCGCTCCCACCACCGTCCGGCCACCGTGCTACAAC
SA-47 / TCCCTACGGCGCTAACCCTCCACACCCGCTGCCCTCCGTCCTCCCTGCCACCGTGCTACAAC
SA-48 / TCCCTACGGCGCTAACCCCTCCTCCAACCGTCCCACCCTGCCACTCGCCACCGTGCTACAAC
SA-49 / TCCCTACGGCGCTAACCACCTCCGCTACCCTGCCAGCCCTCCCCCCGCCACCGTGCTACAAC
SA-50 / TCCCTACGGCGCTAACCCCTCCCGTCCAGTCGTCCTCGCCCCCAACGCCACCGTGCTACAAC
SA-51 / TCCCTACGGCGCTAACCCACCATCCCGGCCAAGCTCCACAAGTCCCGCCACCGTGCTACAAC
SA-52 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-54 / TCCCTACGGCGCTAACCCACTCCCCAGAAGCCTCCACCGAACGCCAGCCACCGTGCTACAAC
SA-55 / TCCCTACGGCGCTAACCCCTCCTCACCCCGCGTCCTCCCACGTCTCGCCACCGTGCTACAAC
SA-56 / TCCCTACGGCGCTAACCGGTTGGGGTGGTGGGGGAGGGCCAGAGGAGCCACCGTGCTACAAC
SA-57 / TCCCTACGGCGCTAACCCTCCCCGCAACACGCACTCCCTGTCTCCCGCCACCGTGCTACAAC
SA-58 / TCCCTACGGCGCTAACCCCCAACGCTCTCCCTGCCCCGCGACGCGAGCCACCGTGCTACAAC
SA-59 / TCCCTACGGCGCTAACCCTCCCCGTCACCGCTCACCACCGTCCTCCGCCACCGTGCTACAAC
SA-60 / TCCCTACGGCGCTAACCCCCCAGCTCTCCCTCCGATCCCAGTCACCGCCACCGTGCTACAAC
SA-61 / TCCCTACGGCGCTAACCTCCCAACCGCTCCACCCTGCCTCCGCCTCGCCACCGTGCTACAAC
SA-62 / TCCCTACGGCGCTAACCCCCCTCGCTCCCGCACACCACCACCGACCGCCACCGTGCTACAAC
SA-63 / TCCCTACGGCGCTAACCCCTCTCCGTCCCCCTCCAGCCAACCTCCGGCCACCGTGCTACAAC
SA-64 / TCCCTACGGCGCTAACCCACCAGCTCTCCCGTCTCCCCCCGCCTCCGCCACCGTGCTACAAC
SA-65 / TCCCTACGGCGCTAACCACCTCCACCCGTCCATCCCCGAACCCTCCGCCACCGTGCTACAAC
SA-66 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-67 / TCCCTACCACAGTGGCGGAGGGGTGAGACTGGTTGCGAGGACCGGGGTTAGCGCCGTAGGGA
SA-68 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-70 / TCCCTACGGCGCTAACCCCCCAGAAACCTCCGCCTCACCGCCACCAGCCACCGTGCTACAAC
SA-71 / TCCCTACGGCGCTAACCCTCCCCGTCACCGCTCACCACCGTCCTCCGCCACCGTGCTACAAC
SA-72 / TCCCTACGGCGCTAACCCTCCTCCTCCACCGCGAACCGTCCCAGATGCCACCGTGCTACAAC
SA-73 / TCCCTACGGCGCTAACTCCCGGAACCCCCATCCCGCTCCACCGCCCGCCACCGTGCTACAAC
SA-74 / TCCCTACGGCGCTAACCCGTCCACTCCCCGCTACCCAGGTCCTCCCGCCACCGTGCTACAAC
SA-75 / TCCCTACGGCGCTAACCCCTCACACAGGCTCTCCTCCGCGACCACCGCCACCGTGCTACAAC
SA-76 / TCCCTACGGCGCTAACCCCTCCACACCGCTCCTCCCAACCGCCTACGCCACCGTGCTACAAC
SA-77 / TCCCTACGGCGCTAACCCCATCGATGCACCCTCGCCTCTCCTAACCGCCACCGTGCTACAAC
SA-78 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-79 / TCCCTACGGCGCTAACCACCTCCACCCCAGCCCAGACGTCGCCTCCGCCACCGTGCTACAAC
SA-80 / TCCCTACGGCGCTAACCACCCTCCTCACCACGTCCCGCCACCACCCGCCACCGTGCTACAAC
SA-81 / TCCCTACGGCGCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCACCGTGCTACAAC
SA-82 / TCCCTACGGCGCTAACCCCAGCTCCTCTCCCAGCCAAGCCACCCGTGCCACCGTGCTACAAC
SA-83 / TCCCTACGGCGCTAACCCCCTCACAACCTCACAAGACCGCCCTCCTGCCACCGTGCTACAAC
SA-84 / TCCCTACGGCGCTAACCCCACCGCTCCTCCACCTCCAGCCGACGCCGCCACCGTGCTACAAC
SA-85 / TCCCTACGGCGCTAACCACTCGTCTCCCCCCATCACCGCTACCCCCGCCACCGTGCTACAAC
SA-86 / TCCCTACGGCGCTAACCACCTCCTCCGCACCTCTCCTACGCCTCCCGCCACCGTGCTACAAC
SA-87 / TCCCTACGGCGCTAACCCGCATCCCTCCGCCCTCCTACCCTCCCCGGCCACCGTGCTACAAC
SA-88 / TCCCTACGGCGCTAACCCCCAGGCTCACCCACCACCGCACCTCTCCGCCACCGTGCTACAAC
SA-89 / TCCCTACGGCGCTAACCCCCACGCTCCCAACCTCCCGTCCTCCCCTGCCACCGTGCTACAAC
SA-90 / TCCCTACGGCGCTAACCCCATCCACGCTCCACCCCCACGTCTCTCCGCCACCGTGCTACAAC
SA-91 / TCCCTACGGCGCTAACCCCACGACTCCTCTGCCAACCCGTCCTGACGCCACCGTGCTACAAC
SA-92 / TCCCTACGGCGCTAACCTCACACCCACCGACCGCTCTCCGCCTCCCGCCACCGTGCTACAAC
SA-93 / TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
SA-94 / TCCCTACGGCGCTAACTCCACCCGCATCCCCCTCGTCCTACCCTCCGCCACCGTGCTACAAC
SA-95 / TCCCTACGGCGCTAACCCCTCCTCCCAGCCTCTCCGCCCTCGAAACGCCACCGTGCTACAAC
SA-96 / TCCCTACGGCGCTAACCCTCCAACCCGTCCACTCCACAACACCCCGGCCACCGTGCTACAAC

Supplementary Table 2.Aptamer sequences in the round 8 pool.